ABSTRACT

A carbapenemase-resistant Klebsiella pneumoniae strain, clone ST258 producing KPC-3, was fully characterized. The entire plasmid content was investigated, thereby identifying plasmids of the IncFIIk (two of them similar to pKPQIL and pKPN3, respectively), IncX, and ColE types, carrying a formidable set of resistance genes against toxic compounds, metals, and antimicrobial drugs and a novel iron(III) uptake system.

TEXT

Nosocomial infection with Klebsiella pneumoniae has a worldwide distribution. The presence of invasive devices, contamination of respiratory support equipment, immunocompromised status, treatment in an intensive care unit or nursing home, and use of antibiotics are factors that increase the likelihood of nosocomial infection. Acquisition of K. pneumoniae has become a major problem in many hospitals because of resistance to multiple antibiotics, the scarcity of therapeutic options left to treat these patients, and the association of invasive infections with disturbingly high mortality rates (6).

Carbapenem resistance in K. pneumoniae strains can arise by the loss or modification of porins (OmpK35 and OmpK36) associated with the production of expanded-spectrum or AmpC β-lactamases or by the acquisition of resistance genes encoding metallo-β-lactamases (MBLs) and nonmetallo-carbapenemases (KPC, GES, or OXA types) (9, 16, 17, 21).

In the last several years, an extremely drug-resistant K. pneumoniae sequence type, 258 (ST258), producing the KPC carbapenemase, has been detected as a nosocomial pathogen around the world, causing epidemics of national and international proportions. K. pneumoniae KPC producers have been described mainly in Israel but in recent studies have been also identified in South and North America and in several European countries, including Italy (2, 7, 10, 13, 17, 18, 22).

The blaKPC-3 gene has been found as part of the 10-kb Tn3-like element Tn4401 (3). Tn4401 has been found on both IncN and IncFIIk plasmids (8, 11). Recently, the pKpQIL plasmid carrying blaKPC-3 from K. pneumoniae ST258 from Israel has been completely sequenced (11) (GenBank accession number GU595196).

Active surveillance of carbapenem-resistant K. pneumoniae and retrospective case-control studies have been performed during the last 4 years (2007 to 2010) at the 1,200-bed Umberto I teaching hospital of Rome, Italy (14). Previous studies demonstrated that a K. pneumoniae ST37 clone was dominant in this hospital, colonizing many patients but also causing severe outbreaks in the intensive care unit (5). This clone showed ertapenem resistance and decreased susceptibility to meropenem and imipenem due to extended-spectrum β-lactamase (ESBL) production, depletion of the OmpK35 porin, and the presence of a mutated OmpK36 protein designated OmpK36V (5).

No carbapenemase-producing K. pneumoniae strains were identified in our hospital until June 2010, when an Italian patient was transferred from another Italian hospital to the laparoscopic surgery ward for an abdominal drainage. From this site, a K. pneumoniae strain was isolated (strain 55873), showing resistance to all antibiotics, including carbapenems (MICs: ertapenem, >8 μg/ml; imipenem, 8 μg/ml; meropenem, >16 μg/ml; determined by the Vitek2 system, AST-N089 card [bioMérieux, Marcy l'Etoile, France], according to the European Committee on Antimicrobial Susceptibility Testing [EUCAST] guidelines), except gentamicin, colistin, and tigecycline. The multilocus sequence typing for this strain was carried out as previously described (4), and the strain was assigned to ST258 (http://www.pasteur.fr/recherche/genopole/PF8/mlst/).

Combined-disc tests of meropenem (10 μg) with or without 400 μg of phenylboronic acid (PBA) or 10 μl of 0.1 M EDTA on Mueller-Hinton agar II detected nonmetallo-carbapenemase production in this ST258 strain (19). PCR amplification of β-lactamase genes and sequencing of the amplicons demonstrated the presence of the blaKPC-3, blaTEM-1, and blaSHV-11 genes (1, 5, 12, 15).

The coding sequences of ompK36 and ompK35 genes were amplified and sequenced for the ST258 strain 55873 (5). The ompK35 gene showed a TGA nonsense mutation at codon 89, resulting in early termination of translation and depletion of this porin (data not shown). The ompK36 gene encoded a novel OmpK36 protein variant, showing the insertion of codons Asp135 and Gly136 in the L3 loop of this protein. This insertion, in analogy with what was previously hypothesized for the CTX-M-15-ST37 clone, may contribute to increasing the MICs of different drugs (5, 9). The complete DNA sequences of plasmids contained in this strain were obtained by applying the 454-Genome Sequencer FLX procedure on a library constructed on total plasmid DNA purified by the Invitrogen PureLink HiPure Plasmid Filter Midiprep kit (Invitrogen, Milan, Italy), according to the manufacturer's procedure. Seventy-two contigs ranging from 49,686 to 353 bp, with at least 15-fold coverage, were obtained using the GS-FLX gAssembler software and compared with GenBank data using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). DNA comparison showed the presence of four major plasmid scaffolds, highly homologous to plasmid pKpQIL (NC_014016), pKPN3 (CP000648), 15S (FJ223606), and IncX1 (HM114226). Following the read-status output and BLAST results, contigs were assembled in four complete plasmid sequences by PCR-based gap closure (Fig. 1). In particular, the assembly of contigs flanked by IS26 elements was individually confirmed by PCR and DNA sequencing of the amplicons.

Major structural features of plasmids pKpQIL-IT (JN233705), pKPN-IT (JN233704), IncX-SHV (JN247852), and ColEST258 (JN247853). Plasmids sequenced in this study were compared with plasmids pKpQIL (NC_014016), pKPN3 (CP000648), pX1.0 (HM114226), and 15S (FJ223606), respectively. White boxes indicate plasmid scaffold regions. The genes associated with the Tra locus are indicated as capital letters within the respective boxes. Resistance genes are indicated by orange boxes, except for the blaKPC and blaSHV-11 genes, which are indicated by dark blue boxes. Transposon-related genes (tnpA, tnpR, and tnpM), insertion sequences, and the class1 integrase genes are indicated by light blue boxes. The two putative virulence clusters acquired by the pKPN-IT plasmid with respect to pKPN3 are indicated by red boxes.

An IncFIIk-FIBk plasmid (JN233705; IncF replicons were defined as in reference 20) was very similar to plasmid pKpQIL (96% query coverage, 99% maximum nucleotide identity). The unique significant difference was the composite transposon IS26-aphA1-IS26, conferring kanamycin resistance that the pKpQIL-IT plasmid has acquired, while it was absent on the pKpQIL plasmid from Israel (11). The ST258 strain 55873 also contained another IncFIIk-FIB-like plasmid, which we named pKPN-IT (JN233704), which was highly related to plasmid pKPN3 (79% query coverage, 100% maximum nucleotide identity) identified in K. pneumoniae from the United States, conferring resistance to arsenic, copper, and silver. The plasmid pKPN-IT showed the same scaffold and resistance loci as did pKPN3, but it acquired a Fec-like iron(III) dicitrate transport system, a glutathione ABC transport system, a class 1 integron carrying trimethoprim and streptomycin resistance genes (dfrA12, orfF, and aadA2), and the chloramphenicol and macrolide resistance genes [catA1, mph(A)] (Fig. 1). The presence of these two plasmids endowed this ST258 strain with a formidable set of resistance genes against toxic compounds, metals, and antimicrobial drugs. The presence of the iron(III) uptake system is an interesting feature, likely involved in the capacity of the bacterium to acquire iron in the human host. The third plasmid identified in ST258 strain 55873 (IncX-SHV; JN247852) carried the blaSHV-11 gene and was related to the IncX family of plasmids such as the Escherichia coli plasmid pOLA52 (EU370913; 35% query coverage, 99% maximum nucleotide identity) and the synthetic conjugative molecular parasite pX1.0 (HM114226; 32% query coverage, 96% maximum nucleotide identity). Finally, a small ColE-like plasmid (JN247853), named ColEST258 and carrying the aac(6′)-Ib gene as part of a Tn1331 element, was also identified in this strain (Fig. 1). Interestingly, ColEST258 was identical (100% query coverage, 100% maximum nucleotide identity) to the K. pneumoniae plasmid 15S from the United States that contained the Tn4401 transposon carrying the blaKPC-2 gene variant (8).

Our study contributes to the description of the characteristics of K. pneumoniae clones that currently represent a serious potential risk for nosocomial settings.